The proliferation of mammalian cells is driven by the core cell cycle machinery. The key components of this machinery are proteins called cyclins, which bind, activate and provide substrate specificity to the cyclin- dependent kinases (CDKs). Cyclin-CDK complexes phosphorylate cellular proteins, thereby driving cell proliferation. Cyclins operating during the G1 phase of the cell cycle (the so-called G1 cyclins) are of particular importance to the cancer field, as many oncogenic pathways were shown to impinge on these proteins. Two classes of G1 cyclins operate in mammalian cells: D-type (cyclins D1, D2 and D3) and E-type (E1 and E2). D-cyclins activate CDK4 and CDK6, whereas E-cyclins activate mostly CDK2. Cyclin D-CDK4, D-CDK6 and E-CDK2 complexes phosphorylate an overlapping set of proteins; cyclin E-associated kinase is thought to target a broader spectrum of substrates. In the past, our laboratory generated and characterized mice lacking D-type or E-type cyclins. Although these animals displayed focused abnormalities, the majority of cell types proliferated normally or nearly normally in the absence of D-type or E-type cyclins. We ascribed the absence of more profound phenotypes to the overlapping functions of the two G1 cyclin classes. To better understand the in vivo functions of G1 cyclins, we generated conditional quintuple knockout mouse embryonic stem cells. These cells allow us to study the consequences of an acute shutdown of all five G1 cyclins in embryonic stem cells. In the proposed work, we will use this experimental system to study the molecular functions of G1 cyclins. An important part of our research plan are analyses of G1 cyclin functions in mouse and in human cancer cells. We will focus on glioblastoma, the most aggressive and incurable brain cancer. We will investigate the molecular functions of G1 cyclins in this tumor type, using a mouse model of glioblastoma as well as primary patient-derived glioblastoma cells. We will test whether inhibition of G1 cyclin function might represent a therapeutic strategy in this deadly cancer type. We will address the following major issues in our three Specific Aims: In Specific Aim 1, we will study the function of G1 cyclins in embryonic stem cells. In Specific Aim 2, we will investigate the molecular functions of G1 cyclins during differentiation. Lastly, in Specific Aim 3, we will extend our studies to mous and human patient-derived cancer cells, by analyzing the function of G1 cyclins in glioblastoma cells. Our work may suggest novel therapeutic strategies for treatment of glioblastoma. This tumor represents the most aggressive and incurable brain cancer, and the median survival time of the affected patients is currently less than one year.